title: Cloning, expression, and immunogenicity of novel fusion protein of Mycobacterium tuberculosis based on ESAT-6 and truncated C-terminal fragment of HSP70.
abstract: Tuberculosis (TB) remains as a major public health problem worldwide. Identification and selection of immunodominant antigens of Mycobacterium tuberculosis (MTB), capable of efficiently inducing a protective immune response is the ultimate goal of TB vaccine development studies. Accordingly, this study was designed to produce a novel M. tuberculosis fusion protein consisted of MTB ESAT-6 (early secreted antigenic target-6 kDa), as a potent immunogenic protein, fused to C-terminus of MTB HSP70 (HSP70(359-610)), as an appropriate carrier and adjuvant. The constructed gene was inserted into a prokaryotic expression vector (pQE30); consequently, the recombinant fusion protein with a 6xHis-tag was successfully over expressed in Escherichia coli M15. Inclusion bodies from bacterial cell lysates were solubilized and the recombinant fusion protein was easily purified by Ni-NTA affinity chromatography under denaturing conditions followed by urea gradient dialysis. The purified and refolded protein was then applied for immunization of mice that resulted in the detection of high titers of specific antibodies, high level of IFN-γ and cell proliferation. The results of our study could confirm the capability of E6H70C fusion protein, as a potential tuberculosis vaccine candidate, for the efficient induction of specific immune responses in a mouse model. However, further investigation need to evaluate the protectivity of this recombinant protein in host model.
authors.affiliation: Biotechnology Department, Razi Vaccine and Serum Research Institute, Karaj, Iran. m.tebianian@rvsri.ir
authors.forename: Majid
authors.initials: M
authors.lastname: Tebianian
authors.forename: Ahmad Zavaran
authors.initials: AZ
authors.lastname: Hoseini
authors.forename: Seyyed Mahmoud
authors.initials: SM
authors.lastname: Ebrahimi
authors.forename: Arash
authors.initials: A
authors.lastname: Memarnejadian
authors.forename: Ali Rezaei
authors.initials: AR
authors.lastname: Mokarram
authors.forename: Mehdi
authors.initials: M
authors.lastname: Mahdavi
authors.forename: Nooshin
authors.initials: N
authors.lastname: Sohrabi
authors.forename: Morteza
authors.initials: M
authors.lastname: Taghizadeh
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D016207:Cytokines / Q000096:biosynthesis; D004269:DNA, Bacterial / Q000235:genetics; D005260:Female; D015870:Gene Expression; D005798:Genes, Bacterial; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D007114:Immunization; D008213:Lymphocyte Activation; D051379:Mice; D008807:Mice, Inbred BALB C; D009169:Mycobacterium tuberculosis / Q000235:genetics* / Q000276:immunology*; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D032581:Tuberculosis Vaccines / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification; D022223:Vaccines, Subunit / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D016207:Cytokines; D004269:DNA, Bacterial; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D018840:HSP70 Heat-Shock Proteins; C444642:HSP70 protein, Mycobacterium tuberculosis; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D032581:Tuberculosis Vaccines; D022223:Vaccines, Subunit